Assays for IGFBP7 having improved performance in biological samples

ABSTRACT

The invention provides IGFBP7 immunoassays with improved clinical performance, particularly when used in the evaluation of renal injuries. The immunoassays rely on the selection and use of antibodies and antibody pairs that exhibit improved assay performance when used in complex clinical specimens such as biological fluids, and particularly when used in rapid assay formats such as lateral flow test devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 15/035,205, filed May 6, 2016, now U.S. Pat. No. 9,822,172, issuedNov. 21, 2017, which is the U.S. national phase of International PatentApplication No. PCT/US2014/064327, filed Nov. 6, 2014, which designatedthe U.S. and application claims the benefit of U.S. ProvisionalApplication No. 61/900,942, filed Nov. 6, 2013, and to U.S. ProvisionalApplication No. 62/054,324, filed Sep. 23, 2014, and to U.S. ProvisionalApplication No. 62/064,380, filed Oct. 15, 2014, each of which is herebyincorporated in its entirety including all tables, figures, and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 20, 2017, isnamed ASTM_0004_CT_SeqListing.txt and is 9 kilobytes in size.

BACKGROUND

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

IGFBP7 (human precursor Swiss Prot entry Q16270) is a secreted proteinwhich is involved in regulation of of insulin-like growth factorexpression in tissue and which modulates IGF binding to its receptors.It also reportedly stimulates prostacyclin production and cell adhesion.IGFBP7 suppresses growth and colony formation of prostate and breastcancer cell lines through an IGF independent mechanism, which causes adelay in the G1 phase of the cell cycle, and increased apoptosis. IGFBP7is expressed in a wide range of normal human tissues and it usuallyshows reduced expression in cancer cell lines of prostate, breast,colon, and lung origin.

In addition, WO2011/097539 and WO2011/075744, each of which is herebyincorporated by reference in its entirety including all tables, figuresand claims, describe the use of IGFBP7 for evaluating the renal statusof a subject both individually and in multimarker panels. In particular,IGFBP7 levels measured by immunoassay are shown to correlate to riskstratification, diagnosis, staging, prognosis, classifying andmonitoring of the renal status.

Signals obtained from specific binding assays such as immunoassays are adirect result of complexes formed between one or more binding species(e.g., antibodies) and the target biomolecule (i.e., the analyte) andpolypeptides containing the necessary epitope(s) to which the antibodiesbind. Immunoassays are often able to “detect” an analyte; but because anantibody epitope is on the order of 8 amino acids, an immunoassayconfigured to detect a marker of interest will also detect polypeptidesrelated to the marker sequence, so long as those polypeptides containthe epitope(s) necessary to bind to the antibody or antibodies used inthe assay. While such assays may detect the full length biomarker andthe assay result be expressed as a concentration of a biomarker ofinterest, the signal from the assay is actually a result of all such“immunoreactive” polypeptides present in the sample. Such binding assaysmay also detect immunoreactive polypeptides present in a biologicalsample that are complexed to additional species, such as bindingproteins, receptors, heparin, lipids, sugars, etc., provided that thoseadditional species do not interfere in binding between the bindingspecies and the target biomolecule. Typically, however, specific bindingassays are formulated using purified analyte, and complex formation andfragmentation patterns are not considered. This is particularly truewhere the identity of such additional binding species are unknown.

SUMMARY

It is an object of the invention to provide IGFBP7 immunoassays withimproved clinical performance, particularly when used in the evaluationof renal injuries. Specifically, we describe the selection and use ofantibodies and antibody pairs that exhibit improved assay performancewhen used in complex clinical specimens such as biological fluids, andparticularly when used in rapid assay formats.

In a first aspect, the present invention relates to a monoclonalantibody which specifically binds human IGFBP7 and is suitable for usein a sandwich immunoassay. The antibody specifically binds to apolypeptide consisting of LIWNKVKRGHYGVQRTELL PGDRDNL (SEQ ID NO: 1) orSSSSSDTCGPCEPASCPPLP (SEQ ID NO: 2).

In a related aspect, the present invention relates to an antibody pairwhich specifically binds human IGFBP7 and is suitable for use in asandwich immunoassay, the antibody pair comprising a first monoclonalantibody which specifically binds to a polypeptide consisting ofLIWNKVKRGHYGVQRTELLPGDRDNL (SEQ ID NO: 1) and a second monoclonalantibody which specifically binds to a polypeptide consisting ofSSSSSDTCGPCEPASCPPLP (SEQ ID NO: 2).

In another related aspect, the present invention relates to a monoclonalantibody which specifically binds human IGFBP7 and is suitable for usein a sandwich immunoassay. The antibody specifically binds to aconformational epitope of IGFBP7. Conformational epitopes are formed byresidues that are sequentially discontinuous but close together inthree-dimensional space in the IGFBP7 protein. An example of such anantibody, referred to as 1D6, is described below.

In a related aspect, the present invention relates to an antibody pairwhich specifically binds human IGFBP7 and is suitable for use in asandwich immunoassay, the antibody pair comprising a first monoclonalantibody which specifically binds to a conformational epitope of IGFBP7,and a second monoclonal antibody which specifically binds to apolypeptide consisting of LIWNKVKRGHYGVQRTELLPGDRDNL (SEQ ID NO: 1) orSSSSSDTCGPCEPASCPPLP (SEQ ID NO: 2).

In certain embodiments, an antibody of the present invention comprisesone or both of (i) a light chain variable region having an amino acidsequence of SEQ ID NO: 9 or a sequence at least 90% identical to SEQ IDNO: 9, and (ii) a heavy chain variable region having an amino acidsequence of SEQ ID NO: 10 or a sequence at least 90% identical to SEQ IDNO: 10, wherein the antibody specifically binds human IGFBP7. Inpreferred embodiments, the antibody is that which is referred to hereinas IC9E4.1.

In other embodiments, an antibody of the present invention comprises oneor both of (i) a light chain variable region having an amino acidsequence of SEQ ID NO: 11 or a sequence at least 90% identical to SEQ IDNO: 11, and (ii) a heavy chain variable region having an amino acidsequence of SEQ ID NO: 12 or a sequence at least 90% identical to SEQ IDNO: 12, wherein the antibody specifically binds human IGFBP7. Inpreferred embodiments, the antibody is that which is referred to hereinas 1D6.

In certain embodiments, an antibody pair of the present inventioncomprises (i) a first antibody which comprises one or both of (i) alight chain variable region having an amino acid sequence of SEQ ID NO:11 or a sequence at least 90% identical to SEQ ID NO: 11, and (ii) aheavy chain variable region having an amino acid sequence of SEQ ID NO:12 or a sequence at least 90% identical to SEQ ID NO: 12, wherein theantibody specifically binds human IGFBP7; and (ii) a second antibodywhich comprises one or both of (i) a light chain variable region havingan amino acid sequence of SEQ ID NO: 9 or a sequence at least 90%identical to SEQ ID NO: 9, and (ii) a heavy chain variable region havingan amino acid sequence of SEQ ID NO: 10 or a sequence at least 90%identical to SEQ ID NO: 10, wherein the antibody specifically bindshuman IGFBP7. In preferred embodiments, the antibody pair comprises afirst antibody referred to herein as 1D6 and a second antibody referredto herein as IC9E4.1.

The phrase “specifically binds to a polypeptide consisting of” aparticular sequence as used herein is not intended to mean that theantibody does not bind to a longer polypeptide comprising the sequence,or to a shorter polypeptide that is a subset of the sequence. Rather,this phrase is simply intended to mean that the antibody will bind tothe particular recited polypeptide.

Antibodies for use in the claimed methods may be obtained from a varietyof species. For example, the antibodies of the present invention maycomprise immunoglobulin sequences which are rabbit, mouse, rat, guineapig, chicken, goat, sheep, donkey, human, llama or camelid sequences, orcombinations of such sequences (so-called chimeric antibodies).Antibodies for use in the present invention may be identified by theirperformance in immunoassays, and then further characterized by epitopemapping in order to understand the epitopes which are relevant to thatperformance.

Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. Preferably, anepitope for each antibody is contained within SEQ ID NO: 1 or SEQ ID NO:2, which is a sequence obtained from the human IGFBP7 sequence. Incertain embodiments, the first monoclonal antibody comprises at leastone, and preferably 2, 3, or 4 consecutive “critical residues” forbinding to IGFBP7. A “critical residue” is defined as an amino acid ofSEQ ID NO: 1 (or SEQ ID NO: 2) that, when changed to an alanine, reducesbinding of an antibody by at least 50%, and more preferably at least75%, relative to its binding to SEQ ID NO: 1 (or SEQ ID NO: 2) itself.In preferred embodiments, the at least one critical residue is at leastone residue in the sequence TELLPGDRD (SEQ ID NO: 3) or at least oneresidue in the sequence EPASC (SEQ ID NO: 4).

Such monoclonal antibodies may be conjugated to a signal developmentelement or immobilized on a solid support. In an example of a sandwichassay, a first antibody (detectably labeled) and a second antibody(immobilized at a predetermined zone of a test device) form sandwichcomplexes with IGFBP7 in the sample at a predetermined zone of a testdevice. In sandwich assays, the first and second antibodies can be thesame (particularly when polyclonal antibodies are used) or different.Thus, the antibodies of the invention may be used in sandwich pairs, ormay be used individually with another binding entity which is not amonoclonal antibody such as a polyclonal antibody or an aptamer.

The antibodies of the present invention can be used as reagents in testkits for detecting IGFBP7 in biological samples. Such a test kit may,for example, comprise a disposable test device configured to generate adetectable signal related to the present or amount of human IGFBP7 in abiological sample. Alternatively, such a test kit may be formulated forperforming an assay in a clinical analyzer which does not utilize adisposable test device. Preferably, the test kit is an in vitrodiagnostic. The term “in vitro diagnostic” as used herein refers to amedical device which is a reagent, reagent product, calibrator, controlmaterial, kit, instrument, apparatus, equipment, or system, whether usedalone or in combination, intended by the manufacturer to be used invitro for the examination of specimens, including blood and tissuedonations, derived from the human body, solely or principally for thepurpose of providing information concerning a physiological orpathological state, or concerning a congenital abnormality, or todetermine the safety and compatibility with potential recipients, or tomonitor therapeutic measures.

In certain embodiments, the immunoassay is performed in a lateral flowformat. Lateral flow tests are a form of immunoassay in which the testsample flows in a chromatographic fashion along a bibulous ornon-bibulous porous solid substrate. Lateral flow tests can operate aseither competitive or sandwich format assays. Preferred lateral flowdevices are disposable, single use test devices. A sample is applied tothe test device at an application zone and transits the substrate, whereit encounters lines or zones which have been pretreated with an antibodyor antigen. The term “test zone” as used herein refers to a discretelocation on a lateral flow test strip which is interrogated in order togenerate a signal related to the presence or amount of an analyte ofinterest. The detectable signal may be read visually or obtained byinserting the disposable test device into an analytical instrument suchas a reflectometer, a fluorometer, or a transmission photometer. Thislist is not meant to be limiting. Sample may be applied withoutpretreatment to the application zone, or may be premixed with one ormore assay reagents prior to application. In the latter case, theantibody may be provided in a separate container from the disposabletest device.

An antibody of the present invention may be diffusively immobilized to asurface within a disposable test device, such that the antibodydissolves into a sample when the sample contacts the surface. In asandwich assay format, this diffusively bound antibody may bind to itscognate antigen in the sample, and then be immobilized at a detectionzone when the antigen is bound by a second antibody non-diffusivelybound at the detection zone. In a competitive format, its cognateantigen in the sample may compete for binding to the diffusively boundantibody with a labeled antigen provided as an assay reagent.

A kit of the invention can further comprise a calibration to relate thedetectable signal to a concentration of IGFBP7. By way of example, acalibration curve may be provided on an electronic memory device whichis read by the analytical instrument which receives the disposable testdevice, such as a ROM chip, a flash drive, an RFID tag, etc.Alternatively, the calibration curve may be provided on an encoded labelwhich is read optically, such as a 2-D bar code, or transmitted via anetwork connection. The analytical instrument can then use thiscalibration curve to relate a detectable signal from an assay into anIGFBP7 concentration.

In certain embodiment, an assay method performed using the antibody pairof the present invention provides a signal related to the present oramount of human IGFBP7 in a biological sample, wherein the minimumdetectable concentration of IGFBP7 in the assay method is 20 ng/mL orless, more preferably 10 ng/mL or less, 5 ng/mL or less, 1 ng/mL orless, and most preferably 0.1 ng/mL or less.

In related aspects, the present invention provides methods fordetermining the presence or amount of human IGFBP7 in a biologicalsample, comprising:

performing an immunoassay on the biological sample with a firstmonoclonal antibody and a second monoclonal antibody which together formsandwich complexes with human IGFBP7, wherein the immunoassay provides adetectable signal that is related to the presence or amount of humanIGFBP7 in the biological sample bound in the sandwich complexes; andrelating the detectable signal to the presence or amount of human IGFBP7in the biological sample. Preferably, the minimum detectableconcentration of IGFBP7 in the immunoassay is 20 ng/mL or less, morepreferably 10 ng/mL or less, 5 ng/mL or less, 1 ng/mL or less, and mostpreferably 0.1 ng/mL or less.

In particularly preferred embodiments, the first monoclonal antibodybinds to a polypeptide consisting of SEQ ID NO: 1, and the secondmonoclonal antibody binds to the polypeptide consisting of SEQ ID NO: 2,in each case with an affinity of at least 10⁸ M⁻¹.

Preferred assay methods comprise performing an immunoassay that detectshuman IGFBP7. Such immunoassays may comprise contacting said body fluidsample with an antibody that detects the marker, and detecting bindingto that antibody. Preferably, the body fluid sample is selected from thegroup consisting of urine, saliva, blood, serum, and plasma, and mostpreferably urine.

With regard to the antibodies of the present invention, the inventionalso relates to nucleic acids encoding such antibodies, andantibody-expressing cell lines expressing such antibodies in additionalaspects.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the disclosure will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

Definitions

As used herein, the terms “Insulin-like growth factor-binding protein 7”and “IGFBP7” refer to one or more polypeptides present in a biologicalsample that are derived from the Insulin-like growth factor-bindingprotein 7 precursor (Swiss-Prot Q16270 (SEQ ID NO: 5))

        10         20         30         40MERPSLRALL LGAAGLLLLL LPLSSSSSSD TCGPCEPASC        50         60         70         80PPLPPLGCLL GETRDACGCC PMCARGEGEP CGGGGAGRGY        90        100        110        120CAPGMECVKS RKRRKGKAGA AAGGPGVSGV CVCKSRYPVC       130        140        150        160GSDGTTYPSG CQLRAASQRA ESRGEKAITQ VSKGTCEQGP       170        180        190        200SIVTPPKDIW NVTGAQVYLS CEVIGIPTPV LIWNKVKRGH       210        220        230        240YGVQRTELLP GDRDNLAIQT RGGPEKHEVT GWVLVSPLSK       250        260        270        280EDAGEYECHA SNSQGQASAS AKITVVDALH EIPVKKGEGA EL

The following domains have been identified in Insulin-like growthfactor-binding protein 7:

Residues Length Domain ID 1-26 26 Signal peptide 27-282 256 Insulin-likegrowth factor-binding protein 7

Unless specifically noted otherwise herein, the definitions of the termsused are standard definitions used in the art of pharmaceuticalsciences. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apharmaceutical carrier” includes mixtures of two or more such carriers,and the like.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may beobtained from a subject, or may be obtained from biological materialsintended to be provided to the subject. For example, a sample may beobtained from a kidney being evaluated for possible transplantation intoa subject, and an analyte measurement used to evaluate the kidney forpreexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of a subject of interest, such as a patient or transplantdonor. In certain embodiments, such a sample may be obtained for thepurpose of determining the outcome of an ongoing condition or the effectof a treatment regimen on a condition. Preferred body fluid samplesinclude blood, serum, plasma, cerebrospinal fluid, urine, saliva,sputum, and pleural effusions. In addition, one of skill in the artwould realize that certain body fluid samples would be more readilyanalyzed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for a kidney injury marker of the present invention, optionally togetherwith other clinical characteristics, to arrive at a diagnosis (that is,the occurrence or nonoccurrence) of an acute renal injury or ARF for thesubject from which a sample was obtained and assayed. That such adiagnosis is “determined” is not meant to imply that the diagnosis is100% accurate. Many biomarkers are indicative of multiple conditions.The skilled clinician does not use biomarker results in an informationalvacuum, but rather test results are used together with other clinicalindicia to arrive at a diagnosis. Thus, a measured biomarker level onone side of a predetermined diagnostic threshold indicates a greaterlikelihood of the occurrence of disease in the subject relative to ameasured level on the other side of the predetermined diagnosticthreshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity (e.g., worsening renal function, future ARF, ordeath) is referred to as being “indicative of an increased likelihood”of an adverse outcome in a patient.

The term “lateral flow” as used herein refers to flow of reagents in alongitudinal direction through a substantially flat porous material.Such porous material is “substantially flat” if the thickness of thematerial is no more than 10% of the length and width dimensions.

The term “downstream region” as used herein relative to a first regionof a device refers to which receives fluid flow after that fluid hasalready reached the first region.

The term “sample application region” as used herein refers to a portionof an assay device into which a fluid sample of interest is introducedfor purposes of determining a component thereof.

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include those developed and/or usedas solid phases in solid phase binding assays. Examples of suitablesolid phases include membrane filters, cellulose-based papers, beads(including polymeric, latex and paramagnetic particles), glass, siliconwafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels,SPOCC gels, and multiple-well plates. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot. Antibodies or otherpolypeptides may be bound to specific zones of assay devices either byconjugating directly to an assay device surface, or by indirect binding.In an example of the later case, antibodies or other polypeptides may beimmobilized on particles or other solid supports, and that solid supportimmobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described marker. The kit comprises reagents for the analysis ofat least one test sample which comprise at least one antibody thatspecifically binds to the marker. The kit can also include devices andinstructions for performing one or more of the diagnostic and/orprognostic correlations described herein. Preferred kits will comprisean antibody pair for performing a sandwich assay, or a labeled speciesfor performing a competitive assay, for the analyte. Preferably, anantibody pair comprises a first antibody conjugated to a solid phase anda second antibody conjugated to a detectable label, wherein each of thefirst and second antibodies that bind a kidney injury marker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

In certain embodiments, the marker assay is performed using a single-usedisposable test device. Such test devices often take the form of lateralflow devices which are now familiar from the common use ofover-the-counter pregnancy tests. Generally, these assay devices have anextended base layer on which a differentiation can be made between asample addition region and an evaluation region. In typical use, thesample is applied to the sample addition region, flows along a liquidtransport path which runs parallel to the base layer, and then flowsinto the evaluation region. A capture reagent is present in theevaluation region, and the captured analyte can be detected by a varietyof protocols to detect visible moieties associated with the capturedanalyte. For example, the assay may produce a visual signal, such ascolor change, fluorescence, luminescence, and the like, when indicatingthe presence or absence of an analyte in a biological sample.

A sample addition region can be provided, for example, in the form of anopen chamber in a housing; in the form of an absorbent pad; etc. Thesample addition region can be a port of various configurations, that is,round, oblong, square and the like or the region can be a trough in thedevice.

A filter element can be placed in, on, or adjacent to the sampleaddition region to filter particulates from the sample, such as toremove or retard blood cells from blood so that plasma can furthertravel through the device. Filtrate can then move into a porous memberfluidly connected to the filter. Suitable filters for removing orretarding cellular material present in blood are well known in the art.See, e.g., U.S. Pat. Nos. 4,477,575; 5,166,051; 6,391,265; and7,125,493, each of which is hereby incorporated by reference in itsentirety. Many suitable materials are known to skilled artisans, and caninclude glass fibers, synthetic resin fibers, membranes of various typesincluding asymmetric membrane filters in which the pore size varies fromabout 65 to about 15 μm, and combinations of such materials. Inaddition, a filter element can comprise one or more chemical substancesto facilitate separation of red blood cells from blood plasma. Examplesof such chemical substances are thrombin, lectins, cationic polymers,antibodies against one or more red blood cell surface antigens and thelike. Such chemical substance(s) which facilitate separation of redblood cells from plasma may be provided in the filter element bycovalent means, nonspecific absorption, etc.

In certain embodiments, a label zone is located downstream of the samplereceiving zone, and contains a diffusively located labeled reagent thatbinds to the analyte of interest or that competes with the analyte ofinterest for binding to a binding species. Alternatively, the label zonecan be eliminated if the labeled reagent is premixed with the sampleprior to application to the sample receiving zone. A detection zone isdisposed downstream of from the label zone, and contains an immobilizedcapture reagent that binds to the analyte of interest.

The optimum pore diameter for the membrane for use in the invention isabout 10 to about 50 μm. The membranes typically are from about 1 mil toabout 15 mils in thickness, typically in the range of from 5 or 10 mils,but may be up to 200 mils and thicker. The membrane may be backed by agenerally water impervious layer, such as a Mylar® polyester film(DuPont Teijin Films). When employed, the backing is generally fastenedto the membrane by an adhesive, such as 3M 444 double-sided adhesivetape. Typically, a water impervious backing is used for membranes of lowthickness. A wide variety of polymers may be used provided that they donot bind nonspecifically to the assay components and do not interferewith flow of the sample. Illustrative polymers include polyethylene,polypropylene, polystyrene and the like. Alternatively, the membrane maybe self supporting. Other non-bibulous membranes, such as polyvinylchloride, polyvinyl acetate, copolymers of vinyl acetate and vinylchloride, polyamide, polycarbonate, polystyrene, and the like, can alsobe used. In various embodiments, the label zone material may bepretreated with a solution that includes blocking and stabilizingagents. Blocking agents include bovine serum albumin (BSA), methylatedBSA, casein, nonfat dry milk. The device can also comprise additionalcomponents, including for example buffering agents, HAMA inhibitors,detergents, salts (e.g., chloride and/or sulfate salts of calcium,magnesium, potassium, etc.), and proteinaceous components (e.g., serumalbumin, gelatin, milk proteins, etc.). This list is not meant to belimiting.

The device may further comprise various control locations which are readto determine that the test device has been run properly. By way ofexample, a procedural control zone may be provided separate from theassay detection zone to verify that the sample flow is as expected. Thecontrol zone is preferably a spatially distinct region at which a signalmay be generated that is indicative of the proper flow of reagents. Theprocedural control zone may contain the analyte of interest, or afragment thereof, to which excess labeled antibody used in the analyteassay can bind. In operation, a labeled reagent binds to the controlzone, even when the analyte of interest is absent from the test sample.The use of a control line is helpful in that appearance of a signal inthe control line indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in the capturezone can be noted. The device may further comprise a negative controlarea. The purpose of this control area is to alert the user that thetest device is not working properly. When working properly, no signal ormark should be visible in the negative control area.

The outer casing or housing of such an assay device may take variousforms. Typically, it will include an elongate casing and may have aplurality of interfitting parts. In a particularly preferred embodiment,the housing includes a top cover and a bottom support. The top covercontains an application aperture and an observation port. In a preferredembodiment, the housing is made of moisture impervious solid material,for example, a plastic material. It is contemplated that a variety ofcommercially available plastics, including, but not limited to, vinyl,nylon, polyvinyl chloride, polypropylene, polystyrene, polyethylene,polycarbonates, polysulfanes, polyesters, urethanes, and epoxies maybeused to construct a housing. The housing may be prepared by conventionalmethodologies, such as standard molding technologies that are well knownand used in the art. The housing may be produced by molding technologieswhich include, but are not limited to, injection molding, compressionmolding, transfer molding, blow molding, extrusion molding, foammolding, and thermoform molding. The aforementioned molding technologiesare well known in the art and so are not discussed in detail herein. Seefor example, Processes And Materials Of Manufacture, Third Edition, R.A. Lindsberg (1983) Allyn and Baron pp. 393-431.

If necessary, the colorimetric, luminescent, or fluorescent intensity ofthe detectable label being employed may be then evaluated with aninstrument that is appropriate to the label. By way of example, afluorometer can be used to detect fluorescent labels; a reflectometercan be used to detect labels which absorb light, etc. The concentrationof the analyte of interest in the samples may be determined bycorrelating the measured response to the amount of analyte in the samplefluid.

Assay Correlations

The terms “correlating” and “relating” as used herein in reference tothe measurement of biomarkers in an assay refers to determining thepresence, or more preferably the amount, of the biomarker in a samplebased on the signal obtained from the assay. Often, this takes the formof comparing a signal generated from a detectable label on one speciesparticipating in the assay to a predetermined standard curve which canbe used to convert the signal to a concentration or threshold amount ofthe biomarker.

The terms “correlating” and “relating” as used herein in reference tothe use of biomarkers for diagnosis or prognosis refers to comparing thepresence or amount of the biomarker(s) in a patient to its presence oramount in persons known to suffer from, or known to be at risk of, agiven condition; or in persons known to be free of a given condition.Often, this takes the form of comparing an assay result in the form of abiomarker concentration to a predetermined threshold selected to beindicative of the occurrence or nonoccurrence of a disease or thelikelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5th percentile ofthe concentration seen in a normal population. Another method may be tolook at serial samples from the same patient, where a prior “baseline”result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold.Receiver Operating Characteristic (“ROC”) arose from the field of signaldetection theory developed during World War II for the analysis of radarimages, and ROC analysis is often used to select a threshold able tobest distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1-specificity, the ROC graph is sometimes called the sensitivity vs(1-specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1-specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1-sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). V_(H) refers to the variabledomain of the heavy chain. V_(L) refers to the variable domain of thelight chain. According to the methods used in this invention, the aminoacid positions assigned to CDRs and FRs may be defined according toKabat (Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991)). Amino acidnumbering of antibodies or antigen binding fragments is also accordingto that of Kabat.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

Ordinarily, an antibody may comprise heavy and/or light chain variablecomprising an amino acid sequence having at least 75% amino acidsequence identity or similarity with the amino acid sequence of eitherthe heavy or light chain variable domain of a parent antibody havingknown binding characteristics, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties) with the species-dependent antibody residues, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence outsideof the variable domain shall be construed as affecting sequence identityor similarity. Naturally occurring residues are divided into groupsbased on common side-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr, asn, gln;-   (3) acidic: asp, glu;-   (4) basic: his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

While conservative substitutions are often preferred, non-conservativesubstitutions (which entail exchanging a member of one of these classesfor a member of another class) are also contemplated.

Preferred therapeutic antibodies are IgG antibodies. The term “IgG” asused herein is meant a polypeptide belonging to the class of antibodiesthat are substantially encoded by a recognized immunoglobulin gammagene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4. In micethis class comprises IgG1, IgG2a, IgG2b, IgG3. The known Ig domains inthe IgG class of antibodies are VH, Cγ1, Cγ2, Cγ3, VL, and CL. IgG isthe preferred class for therapeutic antibodies for several practicalreasons. IgG antibodies are stable, easily purified, and able to bestored under conditions that are practical for pharmaceutical supplychains. In vivo they have a long biological half-life that is not just afunction of their size but is also a result of their interaction withthe so-called Fc receptor (or FcRn). This receptor seems to protect IgGfrom catabolism within cells and recycles it back to the plasma.

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. The light and heavy chainvariable regions show significant sequence diversity between antibodies,and are responsible for binding the target antigen. Each chain is madeup of individual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins.

The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, Preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, K_(on) is the association rate constant andK_(d) is the equilibrium constant). Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor atequilibrium; c=free ligand concentration at equilibrium; K=equilibriumassociation constant; and n=number of ligand binding sites per receptormolecule. By graphical analysis, r/c is plotted on the Y-axis versus ron the X-axis, thus producing a Scatchard plot. Antibody affinitymeasurement by Scatchard analysis is well known in the art. See, e.g.,van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold,Comput. Methods Programs Biomed. 27: 65-8, 1988.

Antibodies of the invention may be further characterized by epitopemapping, so that antibodies and epitopes may be selected that have thegreatest clinical utility in the immunoassays described herein. The term“epitope” refers to an antigenic determinant capable of specific bindingto an antibody. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. Preferably, an epitope is targeted which is present on thetarget molecule, but is partially or totally absent on non-targetmolecules.

In some embodiments, the antibody scaffold can be a mixture of sequencesfrom different species. As such, if the antibody is an antibody, suchantibody may be a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213). The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein). Humanization methods include but are not limited tomethods described in Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, CancerRes.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8. Humanizationor other methods of reducing the immunogenicity of nonhuman antibodyvariable regions may include resurfacing methods, as described forexample in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973.In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759. Other humanization methods mayinvolve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084.

In one embodiment, the antibody is a fully human antibody. “Fully humanantibody” or “complete human antibody” refers to a human antibody havingthe gene sequence of an antibody derived from a human chromosome. Fullyhuman antibodies may be obtained, for example, using transgenic mice(Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or humanantibody libraries coupled with selection methods (Griffiths et al.,1998, Curr Opin Biotechnol 9:102-108).

Production of Antibodies

Monoclonal antibody preparations can be produced using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Monoclonal antibodies derived from animals other than rats and miceoffer unique advantages. Many protein targets relevant to signaltransduction and disease are highly conserved between mice, rats andhumans, and can therefore be recognized as self-antigens by a mouse orrat host, making them less immunogenic. This problem may be avoided whenusing rabbit as a host animal. See, e.g., Rossi et al., Am. J. Clain.Pathol., 124, 295-302, 2005.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Adjuvants that can be used in the methods of antibody generationinclude, but are not limited to, protein adjuvants; bacterial adjuvants,e.g., whole bacteria (BCG, Corynebacterium parvum, Salmonella minnesota)and bacterial components including cell wall skeleton, trehalosedimycolate, monophosphoryl lipid A, methanol extractable residue (MER)of tubercle bacillus, complete or incomplete Freund's adjuvant; viraladjuvants; chemical adjuvants, e.g., aluminum hydroxide, iodoacetate andcholesteryl hemisuccinateor; naked DNA adjuvants. Other adjuvants thatcan be used in the methods of the invention include, Cholera toxin,paropox proteins, MF-59 (Chiron Corporation; See also Bieg et al. (1999)“GAD65 And Insulin B Chain Peptide (9-23) Are Not Primary AutoantigensIn The Type 1 Diabetes Syndrome Of The BB Rat,” Autoimmunity,31(1):15-24, which is incorporated herein by reference), MPL® (CorixaCorporation; See also Lodmell et al. (2000) “DNA Vaccination Of MiceAgainst Rabies Virus: Effects Of The Route Of Vaccination And TheAdjuvant Monophosphoryl Lipid A (MPL),” Vaccine, 18: 1059-1066; Johnsonet al. (1999) “3-O-Desacyl Monophosphoryl Lipid A Derivatives: SynthesisAnd Immunostimulant Activities,” Journal of Medicinal Chemistry, 42:4640-4649; Baldridge et al. (1999) “Monophosphoryl Lipid A (MPL)Formulations For The Next Generation Of Vaccines,” Methods, 19: 103-107,all of which are incorporated herein by reference), RC-529 adjuvant(Corixa Corporation; the lead compound from Corixa's aminoalkylglucosaminide 4-phosphate (AGP) chemical library, see alsowww.corixa.com), and DETOX™ adjuvant (Corixa Corporation; DETOX™adjuvant includes MPL® adjuvant (monophosphoryl lipid A) andmycobacterial cell wall skeleton; See also Eton et al. (1998) “ActiveImmunotherapy With Ultraviolet B-Irradiated Autologous Whole MelanomaCells Plus DETOX In Patients With Metastatic Melanoma,” Clin. CancerRes. 4(3):619-627; and Gupta et al. (1995) “Adjuvants For HumanVaccines—Current Status, Problems And Future Prospects,” Vaccine,13(14): 1263-1276, both of which are incorporated herein by reference).

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

Antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg et al. (1995) “Human Antibodies From TransgenicMice,” Int. Rev. Immunol. 13:65-93, which is incorporated herein byreference in its entirety). For a detailed discussion of this technologyfor producing human antibodies and human monoclonal antibodies andprotocols for producing such antibodies, see, e.g., InternationalPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016,5,545,806, 5,814,318, and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Medarex (Princeton, N.J.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Recombinant Expression of Antibodies

Once a nucleic acid sequence encoding an antibody of the invention hasbeen obtained, the vector for the production of the antibody may beproduced by recombinant DNA technology using techniques well known inthe art. Methods which are well known to those skilled in the art can beused to construct expression vectors containing the antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al, 1990,MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodycan be transferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the antibody of the invention. Inspecific embodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

The host cells used to express the recombinant antibodies of theinvention may be either bacterial cells such as Escherichia coli, or,preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al. (1986) “Powerful And Versatile Enhancer-Promoter UnitFor Mammalian Expression Vectors.” Gene 45:101-105; Cockett et al.(1990) “High Level Expression Of Tissue Inhibitor Of MetalloproteinasesIn Chinese Hamster Ovary Cells Using Glutamine Synthetase GeneAmplification,” Biotechnology 8:662-667).

A variety of host-expression vector systems may be utilized to expressthe antibodies of the invention. Such host-expression systems representvehicles by which the coding sequences of the antibodies may be producedand subsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the antibodies of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells,lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (ratretinal cells developed by Crucell)) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al. (1983) “Easy Identification Of cDNA Clones,” EMBOJ. 2:1791-1794), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye et al. (1985)“Up-Promoter Mutations In The Lpp Gene Of Escherichia coli,” NucleicAcids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of HumanAsparagine Synthetase In Escherichia coli,” J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrixglutathione-agarose beads followed by elution in the presence of freegluta-thione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region Elor E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (see e.g., seeLogan et al. (1984) “Adenovirus Tripartite Leader Sequence EnhancesTranslation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci.(U.S.A.) 81:3655-3659). Specific initiation signals may also be requiredfor efficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al. (1987) “Expression And Secretion Vectors For Yeast,”Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al. (1977)“Transfer Of Purified Herpes Virus Thymidine Kinase Gene To CulturedMouse Cells,” Cell 11:223-232), hypoxanthine-guaninephosphoribosyltransferase (Szybalska et al. (1962) “Genetics Of HumanCess Line. IV. DNA-Mediated Heritable Transformation Of A BiochemicalTrait,” Proc. Natl. Acad. Sci. (U.S.A.) 48:2026-2034), and adeninephosphoribosyltransferase (Lowy et al. (1980) “Isolation Of TransformingDNA: Cloning The Hamster Aprt Gene,” Cell 22:817-823) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) “Transformation Of Mammalian Cells With An AmplfiableDominant-Acting Gene,” Proc. Natl. Acad. Sci. (U.S.A.) 77:3567-3570;O'Hare et al. (1981) “Transformation Of Mouse Fibroblasts ToMethotrexate Resistance By A Recombinant Plasmid Expressing AProkaryotic Dihydrofolate Reductase,” Proc. Natl. Acad. Sci. (U.S.A.)78:1527-1531); gpt, which confers resistance to mycophenolic acid(Mulligan et al. (1981) “Selection For Animal Cells That Express TheEscherichia coli Gene Coding For Xanthine-GuaninePhosphoribosyltransferase,” Proc. Natl. Acad. Sci. (U.S.A.)78:2072-2076); neo, which confers resistance to the aminoglycoside G-418(Tachibana et al. (1991) “Altered Reactivity Of Immunoglobutin ProducedBy Human-Human Hybridoma Cells Transfected By pSV2-Neo Gene,”Cytotechnology 6(3):219-226; Tolstoshev (1993) “Gene Therapy, Concepts,Current Trials And Future Directions,” Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) “The Basic Science Of Gene Therapy,” Science260:926-932; and Morgan et al. (1993) “Human gene therapy,” Ann. Rev.Biochem. 62:191-217). Methods commonly known in the art of recombinantDNA technology which can be used are described in Ausubel et al. (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY;Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds),1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY.;Colbere-Garapin et al. (1981) “A New Dominant Hybrid Selective MarkerFor Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14; and hygro, whichconfers resistance to hygromycin (Santerre et al. (1984) “Expression OfProkaryotic Genes For Hygromycin B And G418 Resistance AsDominant-Selection Markers In Mouse L Cells,” Gene 30:147-156).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel,“The Use Of Vectors Based On Gene Amplification For The Expression OfCloned Genes In Mammaian Cells,” in DNA CLONING, Vol. 3. (AcademicPress, New York, 1987)). When a marker in the vector system expressingan antibody is amplifiable, increase in the level of inhibitor presentin culture of host cell will increase the number of copies of the markergene. Since the amplified region is associated with the nucleotidesequence of the antibody, production of the antibody will also increase(Crouse et al. (1983) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Mol. Cell. Biol. 3:257-266).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot (1986) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Nature 322:562-565; Kohler (1980)“Immunoglobulin Chain Loss In Hybridoma Lines,” Proc. Natl. Acad. Sci.(U.S.A.) 77:2197-2199). The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

Once the antibody of the invention has been recombinantly expressed, itmay be purified by any method known in the art for purification of anantibody, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

EXAMPLES Example 1 Monoclonal Antibody Development in Rabbits

Female New Zealand Rabbits were immunized by subcutaneous injections(SQ) with antigen/adjuvant emulsions. Primary immunization was done withComplete Freund's Adjuvant and Incomplete Freund's Adjuvant was used forall subsequent boosts. Rabbits were injected SQ every three weeks at 250μg protein antigen per rabbit (alternating two sites, hips andscapulas). A test bleed was taken from the marginal ear vein seven daysafter the second boost. This test bleed (immune sera) was tested byindirect ELISA assay to determine if immune response of the rabbit wasadequate for monoclonal antibody development. The best responding rabbitwas given a final SQ boost and four days later was euthanized viaexsanguination. The whole blood was collected via cardiac puncture. Bcells producing antibody of interest were identified by indirect ELISAon target antigen and immunoglobulin genes were isolated. Heavy andlight chains were cloned into separate mammalian expression vectors,transfected into HEK cells (transient transfection), and tissue culturesupernatant containing rabbit monoclonal antibodies were harvested.

Example 2 Monoclonal Antibody Development in Mice

Female BALB/c mice (60 days old) were immunized by intraperitonealinjections (IP) with antigen/adjuvant emulsions as per standardoperating procedure. Primary immunization was done with CompleteFreund's Adjuvant and Incomplete Freund's Adjuvant was used for allsubsequent boosts. Mice were injected IP every 3 weeks at 25 μg antigenper mouse (total volume 125 μL per mouse). Test bleeds were done bysaphenous vein lancing 7 to 10 days after the second boost. This testbleed (immune sera) was tested by indirect ELISA assay to determine ifthe immune response of mice was adequate for fusion. The best 2responding mice were given a final intravenous boost of 10 μg antigenper mouse in sterile saline via lateral tail vein. 4 days after the IVboost the mice were euthanized and the spleens were harvested.Lymphocytes isolated from the spleen were used in the fusion process toproduce hybridomas using the method of Kohler, G.; Milstein, C. (1975).“Continuous cultures of fused cells secreting antibody of predefinedspecificity”. Nature 256 (5517): 495-497. Hybridomas were generatedusing a PEG1500 fusion process.

Example 3 Screening of Antibodies with Patient Samples (Microtiter-basedELISA Method)

-   -   Materials:    -   96-well high bind ELISA plates-Costar 3590 (Corning)    -   ELISA coating buffer: PBS    -   ELISA wash buffer: PBS with 0.02% Tween-20    -   ELISA blocking Buffer (Thermo Pierce, catalogue number N502)    -   ELISA reagent diluent: 200 mM Tris, 1% BSA (BioFx), 0.05%        Tween-20, pH 8.1    -   Neutravidin-HRP conjugate (Thermo Pierce, catalogue number        31001)    -   1-Step Ultra TMB substrate (R&D systems, catalogue number 34028)    -   Stop solution: 2N sulfuric acid    -   Capture antibodies    -   Biotin conjugated detection antibodies    -   Recombinant human IGFBP7 (Peprotech, catalogue number 410-02)    -   EXLx405 plate washer (Biotek)    -   Multiskan FC plate reader (Fisher Scientific)

Testing Procedure

Purified, recombinant IGFBP7 analyte was spiked into Reagent Diluent andserially diluted to generate a set of standard samples covering a rangeof concentrations. Frozen single-use aliquots of patient samples werethawed in a room temperature water bath for 10 minutes, and then dilutedto desired level with Reagent Diluent.

100 μL of 5 μg/mL Capture Antibody solution prepared in coating bufferwas added to each well on a 96-well high bind ELISA plate and incubatedover night at room temperature (22° C. to 25° C.). Each well wasaspirated and washed three times with 300 μL of wash buffer using anautowasher. Then 250 μL of ELISA blocking buffer was added to each well.After an incubation of 2 hours at room temperature, the aspiration/washstep described above was repeated.

100 μL of standard or patient samples was added to each well of theprepared plate and incubated at room temperature on a horizontal orbitalshaker. After 2 hours of incubation, the plate was washed as describedabove. Then 100 μL of 0.1 μg/mL detection antibody solution prepared inreagent diluent was added to each well. After incubation for 1 hour atroom temperature, the plate was washed again. A 0.1 μg/mL solution ofneutravidin-HRP conjugate was prepared in reagent diluent, and 100 μL ofthis solution was added to each well. The plate was incubated for 1 hourat room temperature and washed. 100 μL of 1-step ultra TMB substrate wasadded to each well, incubated at room temperature for 10 minutesprotected from light, followed by 50 μL of stop solution. The opticaldensity in each well was measured with a microplate reader set to awavelength of 450nm.

Example 4 Screening of Antibodies with Patient Samples (Lateral FlowStrip Testing Method)

-   -   Materials:    -   Nitrocellulose membrane    -   Backing card    -   Sample pad    -   Wicking pad    -   Membrane blocking buffer: 10 mM Sodium phosphate, 0.1% sucrose,        0.1% BSA, 0.2% PVP-40, pH 8.0    -   Sample pad blocking buffer: 5 mM Borate, 0.1% Tween-20, 0.25%        PVP-40, 0.5% BSA, pH 8.5    -   Running buffer J: 500 mM Tris, 0.2% 10G, 0.35% Tween-20, 0.25%        PVP-40, pH 8.5    -   Fluorescently-conjugated antibodies    -   Test line antibodies    -   Goat-anti-mouse positive control antibodies    -   Recombinant human IGFBP7

Strip Assembly

Nitrocellulose membranes were striped with test line antibodies using anAD3050 aspirate dispense system, blocked with the membrane blockingbuffer and dried at 37° C. for 30 min. After curing over night in adesiccator, the striped and blocked nitrocellulose membranes werelaminated onto backing cards with wicking pads and sample padspre-treated with the sample pad blocking buffer. The cards were cut into5 mm wide test strips, which were then placed into cartridges.

Sample Preparation

Purified, recombinant IGFBP7 analyte was spiked into Running buffer Jand serially diluted to generate a set of standard samples covering arange of concentrations. Frozen single-use aliquots of patient sampleswere thawed in a room temperature water bath for 10 minutes, and thendiluted to desired level with Running buffer J.

Testing Procedure

10 μL of fluorescently conjugated antibody (0.025 μg/μL) in PBS wasadded to 100 μL of sample. 100 μL of this solution was then loaded intothe input port on the cartridge. Results was read at t=20 minutes usinga fluorescence reader and associated software.

Example 5 Peptide Mapping

Materials: 96-well high bind microtiter plates, Neutravidin,biotinylated peptides, Unconjugated antibodies, mouse IgG, rabbit IgG,goat IgG, HRP conjugated to anti-mouse IgG HRP conjugate, anti-rabbitIgG HRP conjugate, anti-goat IgG HRP conjugate, TMB substrate, 2Nsulfuric acid were used for epitope mapping experiments.

Neutravidin was immobilized in individual wells of 96-well high bindmicrotiter plate. The plates were washed to remove unreacted neutravidinfollowed by a blocking step. Biotinylated peptides were dissolved in anaqueous buffer to a concentration of 10 μg/mL. 50 μL of the peptidesolutions were added to each well of neutravidin coated microtiterplates. These plates were incubated one hour at room temperature andthen washed to remove unbound peptides. Unconjugated mouse and rabbitantibodies were diluted to 5 μg/mL and added to the plate at 100μL/well. Anti-mouse IgG (in the mouse anti-IGFBP7) or anti-rabbit IgG(in the case of rabbit anti-IGFBP7) was added to neighboring wells as anegative control. Plates were incubated 1 hour at room temperature andwashed. HRP conjugated to anti-mouse IgG (in the case of mouseanti-IGFBP7 and mouse IgG negative control), and HRP conjugated toanti-rabbit IgG (in the case of rabbit anti-IGFBP7 and rabbit IgGnegative control) was diluted to 0.2 μg/mL and 100 μL was added to eachwell of the plate. These plates were incubated for 20 minutes at roomtemperature and washed. 100 μL/well of TMB substrate was added andplates were incubated for 20 minutes while avoiding exposure to light.50 μl/well of Stop solution (2N sulfuric acid) was added to each welland plates to stop the reaction. The absorbance was read onspectrophtometric 96-well microplate reader set to measure the opticaldensity at 450 nm.

Example 6 Alanine Scanning Peptide Mapping

Alanine scanning is a widely used mutagenesis approach in which residuesin a target protein are systematically substituted for alanine atselected positions by site-directed mutagenesis, expressed, and assayedfor function. Substitution with alanine residues eliminates side-chaininteractions without altering main-chain conformation or introducingsteric or electrostatic effects. Using automated mutagenesis protocols,every residue in a target polypeptide is changed to alanine, andcritical residues that comprise each antibody binding domain can bedetermined.

Example 7 Results

Using the combined alanine scanning and peptide mapping results, uniqueIGFBP7 monoclonal antibodies were identified and selected based onanalytical performance.

Astute Sequence Antibody Pepscan sequence (total region) 7G2.1₂₁₀PGDRD₂₁₄ ₂₀₁YGVQRTELLPGDRDNL₂₁₆ (SEQ ID NO: 6) (SEQ ID NO: 6) 6D2.1₂₀₆TELLPGDR₂₁₃ ₁₉₁LIWNKVKRGHYGVQRT₂₀₆ (SEQ ID NO: 3) (SEQ ID NO: 7)1C9E4.1 ₃₆EPASC₄₀ ₂₅SSSSSDTCGPCEPASCPPLP₄₄ (SEQ ID NO: 4) (SEQ ID NO: 8)

Example 8 Sequencing Data

Antibody IC9E4.1 was isotyped as a murine IgG1/kappa antibody. cDNA fromthe monocloncal cell line was obtained for sequencing by standardmethods. The sequences of the heavy chain variable region and the lightchain variable region were as follows:

V_(light) (SEQ ID NO: 9)DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL YSNGETYLHW LLQRPGQSPK 50RLIYLVSKLD SGVPDRFTGS GSRTDFTLKI SRVEAEDLGV YYCAQGTHFP 100 HTFGGGTKLEV_(heavy) (SEQ ID NO: 10)QIQLVQSGPE LKKPGETVKI SCKASGYSFT DYSIHWVKQA PGKGLKWMGL 50INTETGEPIY VDDFKGRFAF SLETSARTAY LQINNLKNED TATYFCARAY 100 YWAYWGQGTL V

Antibody 1D6

Antibody 1D6 was isotyped as a murine IgG1/kappa antibody. By epitopemapping, the 1D6 antibody was determined to bind to a conformationalepitope of IGFBP7. cDNA from the monocloncal cell line was obtained forsequencing by standard methods. The sequences of the heavy chainvariable region and the light chain variable region were as follows:

V_(light) (SEQ ID NO: 11)QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMHWYQQKSG TSPKRWIYDT 50SELASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSSPFTFGSG 100 TKLEIKRV_(heavy) (SEQ ID NO: 12)QIQLVQSGPE LKKPGETVKI SCKASGYTFK KYGMNWVKQA PGKGLKWMGW 50INTYTGEPIY ADDFKGRFAF SLETSASTAY LQISNLKNED TATYFCAREEYGPFYAMDYW GQGTSVTVSS

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

The use of “or” herein means “and/or” unless stated otherwise.Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. All patents and publicationsmentioned in the specification are indicative of the levels of those ofordinary skill in the art to which the invention pertains prior to thefiling date of the disclosure. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior disclosure.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. An isolated antibody that binds to a human IGFBP7protein, wherein the antibody comprises three complementaritydetermining regions of a light chain variable region having the aminoacid sequence of SEQ ID NO: 9 and three complementarity determiningregions of a heavy chain variable region having the amino acid sequenceof SEQ ID NO: 10, wherein the complementarity determining regions aredefined according to Kabat.
 2. The antibody according to claim 1,wherein the antibody is a monoclonal antibody.
 3. The isolated antibodyaccording to claim 1, wherein the antibody is conjugated to a signaldevelopment element.
 4. The isolated antibody according to claim 1,wherein the antibody is conjugated to a solid support.
 5. An immunoassaymethod for detecting the presence or amount of human IGFBP7, comprising:contacting a sample with the antibody according to claim 1; detectingbinding of the human IGFBP7 in the sample to the antibody; and relatingthe detected binding to the presence or amount of the human IGFBP7.
 6. Anucleic acid encoding an antibody that binds to a human IGFBP7 protein,wherein the antibody comprises three complementarity determining regionsof a light chain variable region having the amino acid sequence of SEQID NO: 9 and three complementarity determining regions of a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 10, whereinthe complementarity determining regions are defined according to Kabat.7. An antibody-expressing cell line, the cell line expressing anantibody that binds to a human IGFBP7 protein and comprises threecomplementarity determining regions of a light chain variable regionhaving the amino acid sequence of SEQ ID NO: 9 and three complementaritydetermining regions of a heavy chain variable region having the aminoacid sequence of SEQ ID NO: 10, wherein the complementarity determiningregions are defined according to Kabat.
 8. An isolated antibody thatbinds to a human IGFBP7protein, wherein the antibody comprises threecomplementarity determining regions of a light chain variable regionhaving the amino acid sequence of SEQ ID NO: 11 and threecomplementarity determining regions of a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 12, wherein thecomplementarity determining regions are defined according to Kabat. 9.The antibody according to claim 8, wherein the antibody is a monoclonalantibody.
 10. The isolated antibody according to claim 8, wherein theantibody is conjugated to a signal development element.
 11. The isolatedantibody according to claim 8, wherein the antibody is conjugated to asolid support.
 12. An immunoassay method for detecting the presence oramount of human IGFBP7, comprising: contacting a sample with theantibody according to claim 8; detecting binding of the human IGFBP7 inthe sample to the antibody; and relating the detected binding to thepresence or amount of the human IGFBP7.
 13. A nucleic acid encoding anantibody that binds to a human IGFBP7 protein, wherein the antibodycomprises three complementarity determining regions of a light chainvariable region having the amino acid sequence of SEQ ID NO: 11 andthree complementarity determining regions of a heavy chain variableregion having the amino acid sequence of SEQ ID NO: 12, wherein thecomplementarity determining regions are defined according to Kabat. 14.An antibody-expressing cell line, the cell line expressing an antibodythat binds to a human IGFBP7 protein and comprises three complementaritydetermining regions of a light chain variable region having the aminoacid sequence of SEQ ID NO: 11 and three complementarity determiningregions of a heavy chain variable region having the amino acid sequenceof SEQ ID NO: 12, wherein the complementarity determining regions aredefined according to Kabat.